An in vitro collagen perfusion wound biofilm model; with applications for antimicrobial studies and microbial metabolomics

Abstract

BackgroundThe majority of in vitro studies of medically relevant biofilms involve the development of biofilm on an inanimate solid surface. However, infection in vivo consists of biofilm growth on, or suspended within, the semi-solid matrix of the tissue, whereby current models do not effectively simulate the nature of the in vivo environment. This paper describes development of an in vitro method for culturing wound associated microorganisms in a system that combines a semi-solid collagen gel matrix with continuous flow of simulated wound fluid. This enables culture of wound associated reproducible steady state biofilms under conditions that more closely simulate the dynamic wound environment. To demonstrate the use of this model the antimicrobial kinetics of ceftazidime, against both mature and developing Pseudomonas aeruginosa biofilms, was assessed. In addition, we have shown the potential application of this model system for investigating microbial metabolomics by employing selected ion flow tube mass spectrometry (SIFT-MS) to monitor ammonia and hydrogen cyanide production by Pseudomonas aeruginosa biofilms in real-time.ResultsThe collagen wound biofilm model facilitates growth of steady-state reproducible Pseudomonas aeruginosa biofilms under wound like conditions. A maximum biofilm density of 1010 cfu slide− 1 was achieved by 30 h of continuous culture and maintained throughout the remainder of the experiment. Treatment with ceftazidime at a clinically relevant dose resulted in a 1.2–1.6 log reduction in biofilm density at 72 h compared to untreated controls. Treatment resulted in loss of complex biofilm architecture and morphological changes to bacterial cells, visualised using confocal microscopy. When monitoring the biofilms using SIFT-MS, ammonia and hydrogen cyanide levels peaked at 12 h at 2273 ppb (±826.4) and 138 ppb (±49.1) respectively and were detectable throughout experimentation.ConclusionsThe collagen wound biofilm model has been developed to facilitate growth of reproducible biofilms under wound-like conditions. We have successfully used this method to: (1) evaluate antimicrobial efficacy and kinetics, clearly demonstrating the development of antimicrobial tolerance in biofilm cultures; (2) characterise volatile metabolite production by P. aeruginosa biofilms, demonstrating the potential use of this method in metabolomics studies.

Milieu matters: Anin vitrowound milieu to recapitulate key features of, and probe new insights into, polymicrobial biofilms

“…Following injury, the wound bed is bathed in protein-rich exudate, which along with additional host elements, results in a characteristic wound milieu [1,3,4,39]. The composition of wound fluid has been widely reported to resemble that of serum [32,33,40], with several in vitro wound studies using serum to mimic wound conditions [22,27,30,41]. To develop an in vitro wound milieu (IVWM), we used fetal bovine serum (FBS) as the base component, to which relevant host matrix and biochemical factors were added.…”

“…To mimic this, fibronectin was added to the IVWM, to resemble lower concentrations as relevant to the wound milieu [57,[62][63][64]. Given its critical role as an extracellular matrix protein in the wound bed [22,23,27,30,[65][66][67][68], collagen was also included in the IVWM (Table 1). At the concentration of collagen added, the IVWM was in liquid form (not a gel) resembling the wound fluid milieu.…”

“…Luria-Bertani (LB) broth is widely-used to study biofilms under in vitro conditions [20,38,[71][72][73], however, its composition (refined yeast extract and tryptone) poorly mimics the infection state. To recapitulate factors relevant to the wound milieu, several biofilm models incorporate fetal bovine serum (FBS), in varying concentrations, into growth conditions [22,26,74,75]. Given that concentrations of FBS employed across different studies range widely, we chose to use the maximum possible FBS concentration (100%) for comparison.…”

“…Recognizing this, recent studies have incorporated serum or plasma in in vitro growth conditions, to more closely represent the host milieu [22][23][24][25][26][27]. This is relevant given that wound fluid has been shown to resemble the biochemical and nutrient profile of serum [22]. However, the wound milieu is more complex, and includes additional host factors and matrix elements [28].…”

“…However, the wound milieu is more complex, and includes additional host factors and matrix elements [28]. To recapitulate this, clot and matrixbased in vitro wound biofilm models have been developed that more closely mimic in vivo conditions [22,27,[29][30][31]. However, these models are technically demanding, lowthroughput, and poorly compatible with standard biofilm assays.…”